1. Field of the Invention
The present invention relates to the reaction between Limulus lysate and endotoxins produced from Gram-negative bacteria as a means for detecting Gram-negative bacteria. More particularly, the present invention relates to a Limulus lysate of substantially improved sensitivity for detecting smaller amounts of endotoxins prepared by treating a lysate with an inhibitor denaturing or precipitating solvent.
2. Description of the Prior Art
Endotoxins are lipopolysaccharides, which are found in the outermost cell wall layer of Gram-negative bacteria. The toxins are not present in Gram-positive bacteria. Endotoxins are heat stable and therefore not easily destroyed. They also elicit a variety of symptoms in human beings and other animals with a range of severity which is typified by high fever and frequently death. Endotoxins are present in almost everything we eat, drink or touch. It is fortunate indeed that endotoxins are not toxic until they enter the bloodstream where as little as 10.sup.-6 gm of endotoxin can produce toxic manifestations and even death.
Because endotoxins are so common, they present special problems as far as drug manufacturers are concerned in the manufacture of drugs which are injected intravenously. Quality control is essential to ensure that endotoxins are not present in the medicinal agents produced. Currently, the rabbit fever test is used to test for the endotoxins, but the test has many disadvantages in that it is expensive, time-consuming, not specific, and is only qualitative.
Against this background was the discovery by Levin and Bang (Bull Johns Hopkins Hosp. 115, 265-274) that a lysate isolated by the extraction of the amoebocytes from Limulus polyphemus, otherwise known as the horseshoe crab, provides a very sensitive means for detecting the presence of endotoxins in materials. When the lysate contacts an endotoxin containing source, a clot forms which is indicative of the presence of endotoxin. The test has been found to be able to detect as little as 10.sup.-12 gm of endotoxin.
Since the discovery of the ability of Limulus lysate to detect the presence of Gram-negative bacteria or endotoxins, several investigations have been reported concerning the scope of Limulus lysate applicability. Nachum, et al, New England J. of Medicine, 289(18), 931 employed the lysate for the detection of Gram-negative bacterial meningitis. The test was found to be superior to such conventional methods as the gram stain and the nitroblue tetrazolium dye test. Both of the conventional methods present numerous technical difficulties and their sensitivity is not satisfactory. Nachum, et al. also were able to substantiate that the Limulus lysate test is inapplicable to the detection of Gram-positive bacterial meningitis, tuberculous meningitis, aseptic meningitis and meningeal leukemia.
Jorgensen, et al, Applied Microbiology, 26(1), 1973, 38-42 applied the Limulus lysate test for the detection of endotoxin in children suspected of having Gram-negative bacteriuria. The Limulus test was found to be more sensitive than other conventional tests for the detection of Gram-negative bacteria.
Despite the fact that earlier investigative work has shown the Limulus lysate test to be the most effective diagnostic test to date for the detection of endotoxin derived from Gram-negative bacteria, problems exist with the test in that the sensitivity of the test is widely variable. For instance, Levin, New England J. Medicine, 24, 1973(1297-1298), has indicated that because of the variable sensitivity of the Limulus test, problems remain with respect to technique and standardization of the test. Indeed, a detailed study by Jorgensen, et al, Applied Microbiology, 26(1), 1973, 43-48, has shown that the potency of Limulus lysate varies widely depending upon the time of year the hemolymph is obtained from the crab for the production of a lysate. For instance, lysate quality appeared diminished in samples prepared in 1972 compared to samples prepared in 1971. The tests of some lysate batches against endotoxin derived from E. coli showed a maximum sensitivity for detecting a minimum of 1 ng/ml of endotoxin, while other batches were incapable of detecting as much as 10 ng/ml of endotoxin. It was postulated that the observed variability of the lysate tests probably resulted from biological variations within the amoebocytes which occurred at various "seasons" of the year.
It is therefore readily apparent that despite the proven applicability of the Limulus lysate test for the rapid detection of endotoxins, a need continues to exist for a method of continuously obtaining Limulus lysate under conditions such that the variability of the potency of the individual batches can be controlled and the sensitivity increased.